WO2002033093A2 - Molecules de l'aggrecanase - Google Patents

Molecules de l'aggrecanase Download PDF

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Publication number
WO2002033093A2
WO2002033093A2 PCT/US2001/032458 US0132458W WO0233093A2 WO 2002033093 A2 WO2002033093 A2 WO 2002033093A2 US 0132458 W US0132458 W US 0132458W WO 0233093 A2 WO0233093 A2 WO 0233093A2
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WIPO (PCT)
Prior art keywords
aggrecanase
seq
sequence
protein
set forth
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PCT/US2001/032458
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English (en)
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WO2002033093A3 (fr
Inventor
Lisa Anne Racie
Natalie Constance Twine
Michael John Agostino
Neil Michael Wolfman
Elisabeth Ann Morris
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Genetics Institute, Llc
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Priority to AU2002224415A priority Critical patent/AU2002224415A1/en
Publication of WO2002033093A2 publication Critical patent/WO2002033093A2/fr
Publication of WO2002033093A3 publication Critical patent/WO2002033093A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals

Definitions

  • the present invention relates to the discovery of nucleotide sequences encoding
  • the invention further relates to the development of inhibitors of, as well as antibodies to the aggrecanase enzymes. These inhibitors and antibodies may be useful for the treatment
  • Aggrecan is a major extracellular component of articular cartilage. It is a proteoglycan responsible for providing cartilage with its mechanical properties of compressibility and elasticity. The loss of aggrecan has been implicated in the degradation of articular cartilage in arthritic diseases. Osteoarthritis is a debilitating
  • aggrecanase A proteolytic activity termed "aggrecanase" is thought to be responsible for the
  • AD AMTS 4 aggrecanase- 1
  • ADAMTS-11 aggrecanase -2
  • ADAM-TS Thrombospondin type 1 motif
  • ADAM-TS family which are capable of cleaving aggrecan at the
  • the present invention is directed to the identification of aggrecanase protein
  • aggrecanase enzymes and processes for the production of aggrecanases. These enzymes
  • invention further includes compositions comprising these enzymes as well as antibodies
  • the invention includes methods for developing inhibitors of aggrecanase which block the enzyme's proteolytic activity. These inhibitors and
  • antibodies may be used in various assays and therapies for treatment of conditions characterized by the degradation of articular cartilage.
  • the nucleotide sequence of the aggrecanase molecule of the present invention is set forth Figure 1. As described in Example 1 the first 780 base pairs is a partial sequence of aggrecanase of the invention followed by the sequence of HsaOl 1374 deposited in Genbank accession no. AJ011374. The invention further includes equivalent
  • SEQ ID No 4 sets forth the nucleotide sequence for Hsa
  • No. 6 are encoded by HsaO l 1374 representing the second translational frame.
  • invention further includes fragments of the amino acid sequence which encode molecules
  • the human aggrecanase protein or a fragment thereof may be produced by
  • No. 1 substantially free from other proteinaceous materials with which it is co-produced.
  • the DNA sequence further comprises a DNA
  • the invention includes methods for obtaining the full length aggrecanase
  • the method for isolation of the full length sequence involves utilizing the aggrecanase
  • the invention includes methods for obtaining the DNA
  • the present invention may include DNA sequences from other species, which are homologous to the human aggrecanase protein and can be obtained using the human sequence.
  • the present invention may also include functional fragments of the aggrecanase protein, and DNA sequences encoding such functional fragments, as well as functional fragments of other related proteins. The ability of such a fragment to function is determinable by assay of the protein in the biological assays described for the assay of the aggrecanase protein.
  • the aggrecanase proteins of the present invention may be produced by culturing a cell transformed with the DNA sequence of SEQ ID No. 2 ccomprising nucleootide # 1
  • the purified expressed protein is substantially free from other proteinaceous materials with which it is co-produced, as well as from other contaminants.
  • the recovered purified protein is contemplated to exhibit proteolytic aggrecanase activity cleaving aggrecan.
  • the proteins of the invention may be further characterized by the ability to demonstrate aggrecan proteolytic activity in
  • Such assays may involve contacting an aggrecan substrate with the aggrecanase molecule and
  • the invention includes methods for developing inhibitors
  • the method may entail the determination of binding sites based on the three
  • Assays for the inhibitors involve contacting a mixture of aggrecan
  • Another aspect of the invention therefore provides pharmaceutical compositions containing a therapeutically effective amount of aggrecanase inhibitors, in a pharmaceutically acceptable vehicle.
  • compositions of the invention may be used in the treatment of diseases characterized by the degradation of aggrecan and/or an upregulation of aggrecanase.
  • the compositions may be used in the
  • the invention includes methods for treating patients suffering from conditions characterized by a degradation of aggrecan or preventing such conditions.
  • methods entail administering to a patient needing such
  • an effective amount of a composition comprising an aggrecanase inhibitor which inhibits the proteilytic activity of aggrecanase enzymes.
  • Still a further aspect of the invention are DNA sequences coding for expression of
  • Such sequences include the sequence of nucleotides in a 5' to 3'
  • the invention further includes the nucleotide sequences set forth in SEQ ID Nos 2 and 3. Further included in the present invention are DNA sequences which hybridize
  • DNA sequence of Figure lor SEQ ID Nos 2 and 3 under stringent conditions with the DNA sequence of Figure lor SEQ ID Nos 2 and 3 and encode a protein having the ability to cleave aggrecan.
  • Preferred DNA sequences include those which hybridize under stringent conditions [see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982), pages 387 to 389]. It is generally preferred that such DNA sequences encode a polypeptide which is at least about 80% homologous, and more preferably at least about 90% homologous, to the
  • the present invention also includes fragments of the DNA sequence shown in Figure 1 or SEQ ID Nos 2 and 3 which encode a
  • polypeptide which retains the activity of aggrecanase.
  • DNA sequences of the present invention are useful, for example, as probes for
  • present invention includes methods of detecting or diagnosing genetic disorders involving the aggrecanase, or disorders involving cellular, organ or tissue disorders in which
  • aggrecanase is irregularly transcribed or expressed.
  • the DNA sequences may also be
  • a further aspect of the invention includes vectors comprising a DNA sequence as
  • vectors may be employed in a novel process for producing an aggrecanase protein of the invention in which a cell line transformed with a DNA sequence encoding an
  • aggrecanase protein in operative association with an expression control sequence therefor, is cultured in a suitable culture medium and an aggrecanase protein is recovered
  • the vectors may be used in gene therapy applications. In such use, the vectors may be transfected into the cells of a patient ex vivo, and the cells may be reintroduced into a patient. Alternatively, the vectors may be introduced into a patient in vivo through targeted transfection.
  • Such polypeptides are characterized by having an amino acid sequence including the
  • polypeptides include a polypeptide which is at least about 80% homologous, and more
  • amino acid changes are induced by mutagenesis, chemical alteration, or by alteration
  • the purified proteins of the present inventions may be used to generate antibodies, either monoclonal or polyclonal, to aggrecanase and/or other aggrecanase -related
  • the present invention also includes antibodies to aggrecanase or other related proteins.
  • the antibodies may be useful for detection and/or purification of aggrecanase or related proteins, or for inhibiting or preventing the effects of aggrecanase.
  • the aggrecanase of the invention or portions thereof may be utilized to prepare antibodies that specifically bind to aggrecanase.
  • Figure 1 sets forth the nucleotide sequence of the isolated aggrecanase clone
  • the human aggrecanase of the present invention comprises nucleotides # 1 to #
  • the human aggrecanase protein sequence comprises amino acids # 1 to # 242 set forth in SEQ ID No.
  • the aggrecanase proteins of the present invention include polypeptides comprising the amino acid sequence of SEQ ID No.1 and having the ability to cleave aggrecan.
  • the aggrecanase proteins recovered from the culture medium are purified by
  • the isolated and purified proteins may be characterized by the ability to cleave aggrecan substrate.
  • the aggrecanase proteins provided herein also include factors encoded by the sequences similar to those of Figure
  • amino acids with basic side chains such as lysine (Lys or K), arginine (Arg or R) and
  • His or H histidine
  • amino acids with acidic side chains such as aspartic acid (Asp or D)
  • glutamic acid Glu or E
  • amino acids with uncharged polar side chains such as
  • tyrosine (Tyr or Y); and amino acids with nonpolar side chains, such as alanine (Ala or A), glycine (Gly or G), valine (Val or V), leucine (Leu or L), isoleucine (lie or I), proline
  • alanine Al or A
  • glycine Gly or G
  • valine Val or V
  • leucine Leu or L
  • isoleucine lie or I
  • proline amino acids with nonpolar side chains
  • glycosylation recognition sites The asparagine-linked glycosylation recognition sites
  • glycosylation enzymes These tripeptide sequences are either asparagine-X-threonine or
  • glycosylated protein even if the glycosylation sites are left unmodified.
  • the present invention also encompasses the novel DNA sequences, free of
  • hybridization washing conditions for example, OJX SSC, 0.1% SDS at 65°C; see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual). Cold Spring Harbor
  • allelic variations naturally-occurring base changes in the species population which may or may not result in an amino acid change
  • SEQ ID NO. 2 and 3 which are caused by point mutations or by induced modifications (including insertion, deletion, and substitution) to enhance the activity, half-life or
  • Another aspect of the present invention provides a novel method for producing
  • the method of the present invention involves culturing a suitable
  • transformed host cells are cultured and the aggrecanase proteins recovered and purified from the culture medium.
  • the purified proteins are substantially free from other proteins
  • Suitable cells or cell lines may be mammalian cells, such as Chinese hamster ovary cells (CHO).
  • CHO Chinese hamster ovary cells
  • the selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening, product production and purification are known in the art. See, e.g., Gething and Sambrook, Nature. 293:620-625 (1981), or alternatively, Kaufman et al, Mol. Cell. Biol. 5(7): 1750-1759 (1985) or Howley et al, U.S. Patent 4,419,446.
  • Another suitable mammalian cell line, which is described in the accompanying examples, is the monkey COS-1 cell line.
  • the mammalian cell CV-1 may
  • Bacterial cells may also be suitable hosts.
  • E. coli e.g., HB 101 , MC1061 are well-known as host cells in the field of biotechnology.
  • DNA encoding the propeptide of Aggrecanase is generally not necessary.
  • Many strains of yeast cells known to those skilled in the art may also be available
  • insect cells may be utilized as host cells in the method of the present
  • Another aspect of the present invention provides vectors for use in the method of
  • the vectors contain the full novel DNA sequences described above which encode the novel factors of the
  • the vectors contain appropriate expression control sequences permitting expression of the aggrecanase protein sequences.
  • vectors incorporating modified sequences as described above are also embodiments of the present invention.
  • the sequence of Figure 1 or SEQ ID No. 2 and 3 or other sequences encoding aggrecanase proteins could be manipulated to express composite aggrecanase molecules.
  • the present invention includes chimeric DNA molecules encoding an aggrecanase proteion comprising a fragment from Figure 1 or SEQ ID No. 2 and 3 linked in correct reading frame to a DNA sequence encoding another aggrecanase polypeptide.
  • the vectors may be employed in the method of transforming cell lines and contain selected regulatory sequences in operative association with the DNA coding sequences of
  • cleaves aggrecan may be useful for the development of inhibitors of aggrecanase.
  • compositions comprising an aggrecanase inhibitor.
  • the inhibitors may be developed using the aggrecanase in screening assays involving a
  • composition of aggrecan substrate with the inhibitor followed by exposure to aggrecan may be used in the treatment of osteoarthritis and other conditions exhibiting
  • the invention further includes antibodies which can be used to detect aggrecanase and also may be used to inhibit the prooteolytic activity of aggrecanase.
  • the therapeutic methods of the invention includes administering the aggrecanase inhibitor compositions topically, systemically, or locally as an implant or device.
  • the dosage regimen will be determined by the attending physician considering various factors which modify the action of the aggrecanase protein, the site of pathology, the severity of
  • Aggrecanase-1 [Science284: 1664- 1666 (1999)] has at least six domains: signal, propeptide, catalytic domain, disintegrin, tsp and c-terminal.
  • the catalytic domain contains a zinc binding signature region, TAAHELGHVKF and a "MET
  • nucleotide sequence of the aggrecanase of the present invention is a nucleotide sequence of the aggrecanase of the present invention.
  • This human aggrecanase sequence was isolated from a dT-primed cDNA library
  • cDNA was made from human stomach RNA purchased from Clontech.
  • the probe to isolate the aggrecanase of the present invention was generated from the sequence obtained from the database search.
  • the DNA probe was radioactively labelled with 32 P and used to screen the human stomach dT-primed cDNA library, under
  • the human candidate #5 sequence obtained aligns with several EST's in the
  • HsaO l 1374 extends the
  • aggrecanase sequence of the present invention about 2 kB at the 3' end.
  • invention can be lined up to create a sequence that is about 40% homologous to
  • the aggrecanase of the present invention contains the zinc biding region signature and a "MET turn", however is missing the signal and propeptide regions.
  • hsaOl 1374 extends our sequence to cover the disintegrin, tsp and c-terminal spacer. It is with these criteria that candidate #5 is considered a novel Aggrecanase family member.
  • the aggrecanse sequence of the invention can be used to design probes for further screening for full length clones containing the isolated sequence.
  • DNA encoding it is transferred into an appropriate expression vector and introduced into mammalian cells or other preferred eukaryotic or prokaryotic hosts including insect host cell culture systems by conventional genetic engineering techniques.
  • the mammalian expression vector pMT2 CXM is a derivative of p91023(b)
  • Plasmid pMT2 CXM is obtained by EcoRI digestion of pMT2-VWF, which has
  • Plasmid pMT2 DNA can be
  • sequences of pMT2. In addition it inserts the following sequence: 5' PO-CATGGGCAGCTCGAG-3' at nucleotide 1 145. This sequence contains the recognition site for the restriction
  • pMT23 A derivative of pMT2CXM, termed pMT23, contains recognition
  • pMT23 DNA may be prepared by conventional methods.
  • pEMC2 ⁇ 1 derived from pMT21 may also be suitable in practice of the invention.
  • pMT21 is derived from pMT2 which is derived from pMT2-VWF. As described above
  • Plasmid pMT2 DNA can be prepared by conventional methods.
  • pMT21 is derived from pMT2 through the following two modifications. First, 76 bp of the 5' untranslated region of the DHFR cDNA including a stretch of 19 G residues from G/C tailing for cDNA cloning is deleted. In this process, a Xhol site is inserted to obtain the following sequence immediately upstream from DHFR: 5' - CTGCAGGCGAGCCTGAATTCCTCGAGCCATCATG-3'
  • a unique Clal site is introduced by digestion with EcoRV and Xbal, treatment with Klenow fragment of DNA polymerase I, and ligation to a Clal linker (CATCGATG).
  • VAI adenovirus associated RNA
  • pMT21 is digested with
  • This fragment is digested with Taql yielding an Eco RI-Taql fragment of 508
  • protruding end which has the following sequence:
  • This sequence matches the EMC virus leader sequence from nucleotide 763 to 827. It also changes the ATG at position 10 within the EMC virus leader to an ATT and is followed by a Xhol site.
  • This vector contains the SV40 origin of replication and enhancer, the adenovirus major late promoter, a cDNA copy of the majority of the adenovirus tripartite leader
  • adenovirus VA I gene adenovirus VA I gene, DHFR and ⁇ -lactamase markers and an EMC sequence, in appropriate relationships to direct the high level expression of the desired cDNA in
  • vectors may involve modification of the aggrecanase-related
  • aggrecanase cDNA can be modified by removing the non-
  • nucleotides may or may not be replaced by other sequences known to be beneficial for
  • aggrecanase-related proteins can be manipulated to express a mature aggrecanase-related protein by deleting aggrecanase encoding propeptide sequences and replacing them with sequences encoding the complete propeptides of other aggrecanase proteins.
  • the coding sequences could be further manipulated (e.g. ligated to other known linkers or modified by deleting non-
  • the modified aggrecanase-related coding sequence could then be inserted into a known
  • yeast vector could also be constructed employing yeast regulatory sequences for intracellular or extracellular expression of the factors of the
  • yeast cells See, e.g., procedures described in published PCT
  • inventions in mammalian, bacterial, yeast or insect host cell systems may involve the construction of cells containing multiple copies of the heterologous Aggrecanase-related gene.
  • the heterologous gene is linked to an amplifiable marker, e.g. the dihydrofolate
  • DHFR reductase gene for which cells containing increased gene copies can be selected for propagation in increasing concentrations of methotrexate (MTX) according to the procedures of Kaufman and Sha ⁇ , J. Mol. Biol.. 159:601-629 (1982).
  • MTX methotrexate
  • plasmid containing a DNA sequence for ann aggrecanase-related protein of the invention in operative association with other plasmid sequences enabling
  • DHFR expressing transformants are selected for growth in alpha media with dialyzed fetal calf serum, and subsequently
  • MTX selected for amplification by growth in increasing concentrations of MTX (e.g. sequential
  • Aggrecanase polypeptides are provided.
  • the aggrecanase gene of the present invention is cloned into the expression vector pED6 [Kaufman et al., Nucleic Acid Res. 19:44885-4490(1991)].
  • COS and CHO DUKX Bll cells are transiently transfected with the aggrecanase sequence of the invention (+/- co-transfection of PACE on a separate pED6 plasmid) by lipofection (LF2000, Invitrogen).
  • Duplicate transfections are performed for each gene of interest: (a) one for harvesting conditioned media for activity assay and (b) one for 35-S- methionine/cysteine metabolic labeling.
  • methionine/ cysteine (Redivue Pro mix, Amersham). Following 6h incubation at 37°C, conditioned media was harvested and run on SDS-PAGE gels under reducing conditions.
  • Proteins are visualized by autoradiography.
  • the proteins are recovered from the cell culture and purified by isolating the aggrecanase-related proteins from other proteinaceous materials with
  • the purified protein may be assayed in accordance with assays described above. Purification is carried out using
  • Protein analysis is conducted using standard techniques such as SDS-PAGE acrylamide [Laemmli, Nature 227:680 (1970)] stained with silver [Oakley, et al. Anal. Biochem. 105:361 (1980)] and by immunoblot [Towbin, et al. Proc. Natl. Acad. Sci. USA

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Abstract

L'invention concerne des nouvelles protéines de l'aggrécanase et les séquences nucléotidiques codant celles-ci, ainsi que des procédés de préparation de celles-ci. L'invention concerne également des procédés de mise au point d'inhibiteurs des enzymes de l'aggrécanase et des anticorps de telles enzymes, aux fins de traitement d'états caractérisés par la dégradation de l'aggrécane.
PCT/US2001/032458 2000-10-18 2001-10-17 Molecules de l'aggrecanase WO2002033093A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003064597A2 (fr) * 2002-01-25 2003-08-07 Wyeth Molecules d'aggrecanase
US20130041137A1 (en) * 2001-04-25 2013-02-14 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute von Willebrand Factor (vWF) - Cleaving Protease
US11261260B2 (en) 2017-06-02 2022-03-01 Merck Patent Gmbh ADAMTS binding immunoglobulins

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1472364A4 (fr) * 2002-02-05 2005-12-28 Wyeth Corp Molecules d'aggrecanase tronquees
US7118902B2 (en) * 2002-07-29 2006-10-10 Wyeth Modified ADAMTS4 molecules and method of use thereof

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EP1152055A1 (fr) * 2000-04-27 2001-11-07 Pfizer Products Inc. Polypeptides ADAMTS, acides nucléiques les codant et leur utilisations

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EP1152055A1 (fr) * 2000-04-27 2001-11-07 Pfizer Products Inc. Polypeptides ADAMTS, acides nucléiques les codant et leur utilisations

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TORTORELLA M D ET AL: "PURIFICATION AND CLONING OF AGGRECANASE-1: MEMBER OF THE ADAMTS FAMILY OF PROTEINS" SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, US, vol. 284, June 1999 (1999-06), pages 1664-1666, XP002937898 ISSN: 0036-8075 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130041137A1 (en) * 2001-04-25 2013-02-14 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute von Willebrand Factor (vWF) - Cleaving Protease
WO2003064597A2 (fr) * 2002-01-25 2003-08-07 Wyeth Molecules d'aggrecanase
WO2003064597A3 (fr) * 2002-01-25 2004-06-03 Wyeth Corp Molecules d'aggrecanase
US11261260B2 (en) 2017-06-02 2022-03-01 Merck Patent Gmbh ADAMTS binding immunoglobulins

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